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S 

43 

Bulletin  269  August,  1925 


r\o,Zk(j>c\ 


(Eimnrrimtf  Agrtmlfural  lExptrtttttttt  J^tatuw 

Jfottt  BJatmt,  (Emtnwttnti 


Perithecia  of   Thielavia  basicola  Zopf 
in  Culture 

And 

The  Stimulation  of  their  Production 
by  Extracts  from  other  Fungi 


FLORENCE  A.  McCORMICK 


BOTANICAL   DEPARTMENT 


The  Bulletins  of  this  Station  are  mailed  free  to  citizens  of  Connecticut 
who  apply  for  them,  and  to  other  applicants  as  far  as  the  editions  permit. 


CONNECTICUT  AGRICULTURAL  EXPERIMENT  STATION 

OFFICERS  AND  STAFF 
August,  1925. 


BOARD  OF  CONTROL. 

His  Excellency,  John  H.  Trumbull,  ex-officio,  President. 

Charles  R.  Treat,   Vice-President Orange 

George  A.  Hopson,  Secretary Mount  Carmel 

Wm.  L.  Slate,  Jr.,  Director  and  Treasurer New  Haven 

Joseph  W.  Alsop Avon 

Elijah    Rogers Southington 

Edward  C.  Schneider Middletown 

Francis  F.  Lincoln Cheshire 


Administration. 


Chemistry: 
Analytical 
Laboratory. 


Biochemical 

Laboratory. 


Botany. 


Entomology. 


STAFF. 
E.  H.  Jenkins,  Ph.D.,  Director  Emeritus. 

Wm.  L.  Slate,  Jr.,  B.Sc,  Director  and  Treasurer. 
Miss  L.  M.  Brautlecht,  Bookkeeper  and  Librarian. 
Miss  J.  V.  Berger,  Stenographer  and  Bookkeeper. 
Miss  Mary  Bradley,  Secretary. 
G.  E.  Graham,  In  Charge  of  Buildings  and  Grounds. 


I 

\       Assistant  Chemists. 

I 


E.  M.  Bailey,  Ph.D.,  Chemist  in  Charge. 

R.  E.  Andrew,  M.A. 

C.  E.  Shepard 

Owen  L.  Nolan 

Harry  J.  Fisher,  A.B. 

W.  T.  Mathis 

Frank  C.  Sheldon,  Laboratory  Assistant. 

V.  L.  Churchill,  Sampling  Agent. 

Miss  Mabel  Bacon,  Stenographer. 

T.  B.  Osborne,  Ph.D.,  Sc.D.,  Chemist  in  Charge. 


G.  P.  Clinton,  Sc.D.,  Botanist  in  Charge. 

E.  M.  Stoddard,  B.S.,  Pomologist. 

Miss  Florence  A.  McCormick,  Ph.D.,  Pathologist. 

Willis  R.  Hunt,  Ph.D.,  Scientific  Assistant 

A.  D.  McDonnell,  General  Assistant. 
Mrs.  W.  W.  Kelsey,  Secretary. 

W.  E.  Britton,  Ph.D.,  Entomologist  in  Charge;  State  Entomologist 

B.  H.  Walden,  B.Agr.      1 

M.  P.  Zappe,  B.S.  \       Assistant  Entomologists. 

Philip  Garman,  Ph.D.      J 

Roger  B.  Friend,  B.Sc,  Graduate  Assistant. 

John  T.  Ashworth,  Deputy  in  Charge  of  Gipsy  Moth  Work. 

R.  C.  Botsford,  Deputy  in  Charge  of  Mosquito  Elimination. 

Miss  Gladys  M.  Finley,  Stenographer. 


Forestry. 


Walter  O.  Filley,  Forester  in  Charge. 
H.  W.  Hicock,  M.F.,  Assistant  Forester. 
Miss  Pauline  A.  Merchant,  Stenographer. 


Plant  Breeding. 
Soil  Research. 


Donald  F.  Jones,  S.D.,  Geneticist  in  Charge. 
P.  C.  Mangelsdorf,  S.D.,  Assistant  Geneticist. 

M.  F.  Morgan,  M.S.,  Investigator 

George  D.  Scarseth,  B.S.,  Graduate  Assistant. 


Tobaco  Sub-Station      P.  J.  Anderson,  Ph.D.,  Pathologist  in  Charge. 
at  Windsor.  N.  T-  Nelson,  Ph.D.,  Plant  Physiologist. 


The  Wilson  H.  Lee  Co. 


Perithecia  of  Thielavia  basicola  Zopf  in 
Culture  and  the  Stimulation  of  their 
Production  by  Extracts  from  other  Fungi. 

By 

Florence  A.  McCormick. 

During  a  disease  survey  of  tobacco  in  the  summer  of  1920, 
Thielavia  basicola  Zopf  was  found  to  be  unusually  abundant,  the 
heavy  rains  in  the  early  part  of  the  season  favoring  its  development. 
Perithecia  were  plentiful  and,  in  some  cases,  they  were  found  to  be 
deeply  embedded  in  the  tissues  of  the  host.  It,  therefore,  seemed 
worth  while  to  attempt  to  establish  more  definitely  the  connection 
of  the  ascospore  stage  with  the  chlamydospores  and  endoconidia, 
all  three  spore  forms  being  known  by  the  name  Thielavia  basicola 
Zopf,  since  Peglion,  in  1900,  (19)  has  given  the  only  published 
record  of  perithecia  in  artificial  culture.  Aside  from  its  scientific 
interest,  it  is  of  economic  value  to  know  whether  there  are  two 
fungi  or  only  one,  possessing  all  spore  forms,  parasitic  on  its 
agricultural  hosts.  Thielavia  basicola  Zopf  is  a  well  known  para- 
site, chiefly  serious  on  tobacco,  the  violet  and  many  of  the 
Leguminosae.  Johnson  mentions  thirty-nine  hosts  compiled  by 
previous  investigators  and  gives  sixty-six  additional  ones. 

Historical. 

The  literature  cited  below  concerns,  in  the  main,  the  perithecia 
commonly  assumed  to  be  the  perfect  stage  of  the  fungus  producing 
chlamydospores  and  endoconidia  known  as  Thielavia  basicola  Zopf 
and  is  only  a  small  part  of  the  mass  of  publications  bearing  chiefly 
on  the  economic  problems  connected  with  that  fungus. 

In  1850  Berkeley  and  Broome  (2)  found  the  chlamydospores 
at  the  base  of  stems  of  peas  and  Nemophila  auriculata  and  gave 
the  fungus  the  name  of  Torula  basicola  n.  s.  In  1876  Zopf  (33)  pub- 
lished an  article  in  which  he  described  a  fungus  growing  on  Senecio 
elegans  L.  in  the  Botanical  Garden  of  Berlin  and  he  recognized 
the  chlamydospores  as  identical  with  those  described  and  figured 
hy  Berkeley  and  Broome.  In  addition  to  the  chlamydospores,  he 
'described  endoconidia,  ascospores  and  spermatia.  The  last  spore 
form  has  not  been  observed  by  other  workers  and  Zopf  himself 
omitted  it  from  a  later  publication.  Due  to  the  association  of 
perithecia  with  the  chlamydospores  and  endoconidia,  Zopf,  in  his 
publication  of  1876,  changed  the  name,  Torula  basicola,  given  by 
Berkeley  and  Broome,  to  Thielavia  basicola  and  placed  the  fungus 
in  the  Perisporiaceae.  Since  the  date  of  that  publication  the 
fungus  bearing  chlamydospores  and  endoconidia,  noted  above, 
has  also  been  known  as  Thielavia  basicola  Zopf  and  the  perithecia 


540  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    269. 

occasionally  found  with  them  have  been  considered  the  perfect 
stage  of  that  fungus.  In  August,  1876,  Sorokin  (30)  announced 
the  finding  of  a  new  fungus  on  the  roots  of  Cochlearia  armoracia. 
He  described  the  chlamydospores,  but,  not  recognizing  them  as 
belonging  to  the  fungus  found  by  Berkeley  and  Broome  twenty-six 
years  before,  nor  knowing  of  Zopf's  discovery,  named  the  fungus 
Helminthosporium  fragile  sp.  n.  Saccardo  lists  the  fungus  in  three 
different  places  and  under  as  many  different  names.  He  retains 
the  name  for  the  chlamydospore  stage  given  by  Berkeley  and 
Broome  (23) ,  but  he  changed  the  name  Helminthosporium  fragile 
Sorok.,  given  by  Sorokin,  to  Clasterosporium  fragile  (Sorok.) 
Sacc.  (24).  He  also  gives  a  description  of  Thielavia  basicola  Zopf 
(22) ,  including  in  it  perithecia  along  with  the  other  spore  forms. 

In  his  paper  of  1891,  Zopf  (34)  gives  a  detailed  account  of  a 
new  disease  of  lupines  which  he  found  to  be  due  to  Thielavia 
basicola.  He  did  not  make  cultures,  but,  since  perithecia  were 
again  found  associated  with  chlamydospores  and  endoconidia,  he 
still  considered  the  fungus  undoubtedly  an  ascomycete  and 
retained  the  name  Thielavia  basicola.  This  opinion  has  been 
generally  accepted  although  the  relationship  of  perithecia  to 
endoconidia  and  chlamydospores  has  not  been  considered  com- 
pletely established,  since  perithecia  are  not  obtained  in  artificial 
cultures  and  only  infrequently  found  in  nature.  In  classification 
of  fungi,  however,  Thielavia  basicola  Zopf  is  invariably  placed  with 
the  ascomycetes,  on  the  supposition  that  chlamydospores,  endo- 
conidia and  perithecia  belong  to  the  same  fungus,  and  taxonomists 
agree  that  it  is  closely  related  to  the  Aspergillaceae. 

Previous  to  the  report  from  this  Station  in  1921  (14),  Peglion  (19) 
is  the  only  person  who  had  obtained  perithecia  in  a  culture.  He 
found  perithecia  only  once,  these  occurring  after  he  moistened  a 
three  year  old  culture  on  potato  plugs  with  a  6%  tartaric  acid  solu- 
tion. A  week  later  he  discovered  that  perithecia  had  developed. 
Prof.  Thaxter,  1891,  (31),  who  found  this  fungus  on  violet  roots  and 
who  was  the  first  to  report  its  presence  in  America,  did  not  find 
perithecia  and  he  has  never  been  fully  convinced  of  their  relation- 
ship to  the  other  two  spore  forms.  Sorauer  (29),  1895,  does  not 
question  the  relationship.  Oudemans  (18)  considers  Thielavia 
basicola  Zopf  as  belonging  to  the  Perisporiaceae  and  he  illustrates 
only  a  mature  perithecium,  a  mature  ascus  and  a  young  ascogo- 
nium,  omitting  any  reference  to  the  asexual  spores.  Cappelluti- 
Altomare  (4)  found  perithecia  associated  with  chlamydospores  on 
tobacco  roots.  Aderhold  (1)  failed  to  obtain  perithecia  in  his 
cultures  and  did  not  find  them  in  plants  infected  with  that  fungus. 
In  discussing  perithecia  Clinton,  1906,  (5)  says,  "So  far  as  our  own 
observations  go  we  could  not  positively  assert  their  relationship 
to  the  other  spore  forms  of  the  root  rot  fungus,  but  from  their  pres- 
ence and  the  observations  of  others  there  seems  to  be  no  reason 
for  doubting  this  relationship."    In  his  report  of  the  following  year 


PERITHECIA    OF    THIELAVIA    BASICOLA  541 

Dr.  Clinton  (6)  further  says  "During  the  past  year  and  a  half  the 
fungus  has  been  under  observation  in  cultures  with  various  media 
in  an  attempt  to  develop  the  ascospore  stage.  This  has  not  been 
obtained,  though  the  fungus  was  grown  on  tobacco  roots  on  which 
this  stage  occurs  in  nature  in  Connecticut.  Fresh  tobacco  roots 
containing  the  fungus  were  sent  to  Professor  Thaxter,  who  tried 
to  isolate  the  ascospores  by  the  Barber  method  and  obtain  cultures 
directly  from  them — our  cultures  having  come  originally  from  the 
endospores,  or  possibly  from  the  chlamydospores — but  he  was  not 
successful  in  obtaining  such  cultures.  Professor  Thaxter  has,  on 
the  other  hand,  a  culture  of  another  species  of  Thielavia  which 
forms  ascospores,  but  never  the  endospores  and  chlamydospores. 
These  facts  possibly  may  indicate  that  the  ascospore  stage  has  no 
relationship  to  our  fungus,  and  that  it  occurs  on  the  tobacco  roots 
as  a  parasite  of  the  fungus  rather  than  as  a  stage  of  it. ' '  Duggar(8) 
says,  "The  association  of  the  ascosporous  stage  with  the  others  and 
the  apparent  continuity  of  mycelium  are  believed  to  show  genetic 
connection."  Gilbert  (12)  also  accepts  this  hypothesis  and  states 
that  he  was  fortunate  to  obtain  a  perithecium  attached  to  the 
same  hypha  which  bore  an  endoconidiophore,  but  that  he  never 
saw  an  ascus.  Foex  (10)  and  Reddick  (21)  found  violets  infected 
with  Thielavia  basicola  Zopf  and  accepted  the  relationship  of 
perithecia  with  the  other  spore  forms.  Johnson  (15)  considers 
association  of  the  perithecia  with  chlamydospores  in  nature  as  an 
evidence  of  their  relationship.  Peters  (20),  who  did  not  obtain 
perithecia  in  his  cultures  and  only  once  found  them  in  his  investi- 
gations in  the  field,  questions  their  connection  with  the  chlamydo- 
spores and  endoconidia. 

Ferraris  (9)  distinguishes  the  asexual  stage  from  the  ascospore 
stage,  applying  the  name  Thielaviopsis  basicola  (Berk.)  Ferraris  to 
the  fungus  which  bears  the  chlamydospores  and  endoconidia  and 
the  name  Thielavia  basicola  Zopf  to  the  ascospore  stage,  not,  how- 
ever, implying  two  distinct  fungi.  To  simplify  the  nomenclature 
in  this  paper  the  writer  is  following  hereafter  the  terminology  of 
Ferraris  for  these  different  stages. 

Experiments  With  a  Conidum-Chlamydospore  Culture. 

In  August,  1920,  a  culture  of  Thielaviopsis  basicola  (Berk.) 
Ferraris,  culture  No.  396,  was  isolated  from  tobacco  roots  in  which 
there  were  large  numbers  of  perithecia  associated  with  chlamydo- 
spores. Pieces  of  the  infected  roots  were  thoroughly  washed  in 
running  water,  followed  by  sterilized  water,  and  put  on  oat  agar 
containing  some  tobacco  leaf  extract.  An  endoconidium-chlamyd- 
ospore  culture  was  obtained,  but  no  perithecia  were  produced. 

Peglion's  result  indicated  that  nutrition  might  be  a  factor  in 
the  development  of  perithecia.  Repeated  efforts  were  made, 
therefore,  to  induce  the  formation  of  perithecia  by  the  use  of 
various  kinds  of  media,  by  the  provision  of  an  excess  of  food,  by 


542  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    269. 

slow  starvation  and,  finally,  by  partial  starvation  followed  by  an 
abundance  of  food. 

It  was  thought  that  tobacco  extract  might  be  a  favorable  food, 
and  agar  was  made  with  it,  alone  and  in  combination  with  other 
substances.  Sterilized  tobacco  seedlings  were  used  alone  and 
made  up  with  agar  and  soil.  Stock  agars  were  made  with  ground 
peas,  beans,  oats,  corn,  peanuts,  carrots,  potatoes  and  beef  extract. 
These  media  were  used  alone  and  also  in  combination  with  other 
nutrients,  such  as  glucose  and  yeast,  and  a  series  of  cultures, 
grading  from  slightly  acid  to  slightly  alkaline,  was  also  tried. 
Plugs  of  carrot,  potato,  pineapple  and  pea  pods  gave  an  excellent 
growth  of  the  fungus,  but  orange  and  lemon  plugs  gave  little  or 
no  growth.  Transfers  from  luxuriant  cultures  to  water  agar  were 
made,  with  the  hope  that  a  change  from  an  abundance  of  food  to  a 
negligible  quantity  might  be  effective,  and  transfers  were  also 
made  from  one  highly  nutritive  medium  to  another  equally  so, 
but  of  a  different  kind.  With  some  cultures  gelatine  was  substi- 
tuted for  agar.  The  results  of  these  tests  were  strictly  negative, 
not  a  single  culture  showed  a  development  of  perithecia.  Eighteen 
cultures  on  various  media  in  protected  test  tubes,  were  planted 
outdoors  in  the  spring  and  were  left  throughout  the  summer  to 
see  if  the  natural  temperature  of  the  soil  might  be  a  factor.  Here 
again  the  results  were  entirely  negative. 

Perithecia  in  Culture. 

In  view  of  the  negative  results  of  the  nutrition  experiments  it 
seemed  worth  while  to  determine  whether  or  not  perithecia  of 
Thielavia  basicola  Zopf  were  the  product  of  sexual  strains.  It 
was  the  intention  of  the  writer  to  secure  during  the  summer  of 
1921  as  large  a  number  of  strains  as  possible  for  the  purpose  of 
making  mixed  cultures,  but  work  on  tobacco  wildfire  prevented 
making  such  collections.  No  other  culture  in  addition  to  culture 
396  was  procured  until  later  in  the  summer  when  some  violet 
plants,  heavily  infected  with  chlamydospores  of  Thielaviopsis 
basicola  (Berk.)  Ferraris  and  perithecia  of  Thielavia  basicola  Zopf, 
were  sent  to  Dr.  Clinton  from  a  greenhouse  near  Hartford.  The 
writer  visited  the  greenhouse  and  secured  fresh  material  for 
culturing. 

On  August  9th  pieces  of  these  roots  were  cultured  like  the 
tobacco  roots  from  which  culture  396  was  obtained,  put  into  four 
Petri  dishes  and  given  the  culture  number  1351.  On  August  15th 
a  slight  growth  of  Thielaviopsis  basicola  was  perceptible  and 
transfers  were  made  to  three  tubes  of  pea  meal  agar  and  three 
tubes  of  carrot  agar.  Two  days  later  additional  transfers  were 
made  to  seven  tubes  of  carrot  agar.  Nothing  further  was  done 
with  these  cultures  until  October  6th.  On  that  date  one  of  the 
transfers  made  on  pea  meal  agar,  August  15th,  showed  chlamydo- 


PERITHECIA    OF    THIELAVIA    BASICOLA  543 

spores,  endoconidia  and  perithecia.  This  tube  was  so  contaminated 
with  bacteria  that,  although  sixteen  transfers  to  tubes  and  two  to 
Petri  dishes  were  made,  only  a  feeble  growth  was  obtained.  Subse- 
quent transfers  failed  to  restore  it  and  the  culture  was  finally  lost. 
Of  the  seven  transfers  made  on  carrot  agar,  August  17th,  one 
showed  perithecia  as  well  as  chlamydospores  and  endoconidia  and 
the  remaining  six  had  only  chlamdospores  and  endoconidia.  The 
growth  of  these  six  cultures  was  feeble  on  account  of  bacterial 
contamination  and  they,  too,  were  finally  lost.  The  culture  con- 
taining perithecia  along  with  the  two  asexual  spores  was  also 
contaminated  with  bacteria  and  a  fungus,  still  undetermined,  but 
repeated  culturing  freed  it  from  bacteria  and  later  from  the  fungus. 

There  can  be  no  doubt  that  culture  1351,  obtained  from  the 
violet,  is  the  fungus  commonly  known  as  Thielavia  basicola  Zopf. 
As  stated  above,  Professor  Thaxter  (31),  in  1891,  announced  the 
finding  of  this  fungus  on  violets  in  Connecticut.  In  regard  to 
perithecia  he  makes  the  following  statement:  "Zopf  describes  an 
ascosporic  condition  on  which  the  genus  Thielavia  is  founded, 
which  has  not  been  observed  by  the  writer  except  as  a  parasite  on 
other  fungi  (species  of  Isaria)."  Foex  (10)  also  found  Thielavia 
basicola  on  violets  and  he  gives  a  description  of  the  disease  and 
figures  chlamydospores  and  perithecia  with  ascospores.  In  regard 
to  the  relationship  of  perithecia  with  the  other  spore  forms  he  says : 
"Toutefois,  il  est  hors  de  doute,  apres  les  observations  de  Zopf,  que 
ces  conceptacles  appartiement  bien  au  champignon  qui  constitue 
des  endoconidies  et  des  chlamydospores."  In  his  paper  on  violet 
diseases  Reddick  (21)  says:  "The  ascosporic  stage,  mentioned 
above,  does  not  seem  to  play  a  particularly  important  role  in  the 
life  cycle  of  this  organism.  If  it  were  commonly  developed  it 
would  seem  reasonably  certain  that  the  numerous  workers  in  this 
country  and  abroad  would  have  seen  and  recorded  its  occurrence 
more  frequently."  Reddick 's  illustrations  of  infected  violet  plants 
agree  well  with  Fig.  1,  Plate  XXXVII.  The  plants  were  consid- 
erably stunted  and  had  comparatively  small,  yellowish  leaves.  In 
some  cases  the  root  system  was  almost  entirely  decayed,  only  a 
few  lateral  roots  remained  and  the  cortex  of  the  main  root  was 
almost  disintegrated,  in  places  exposing  the  network  of  xylem. 

Sections  of  some  diseased  violet  roots,  collected  from  the  green- 
house mentioned  above,  showed  numerous  chlamydospores  borne 
internally  in  the  cortex  as  well  as  in  the  xylem,  medullary  rays 
and  pith.  Fig.  3,  PI.  XXXVII,  taken  from  such  a  root,  also  shows 
sclerotial  masses  of  mycelium  completely  filling  some  of  the  cells. 
The  chlamydospores  shown  in  this  figure  are  identical  with  those 
found  in  tobacco.  Endoconidia  from  the  violet  are  also  like 
those  in  the  culture  isolated  from  the  second  host.  In  addition 
to  Brierley's  description  (3)  of  endoconidia,  Gilbert  (12)  has  also 
described  both  spore  forms  and  it  is  not  necessary  to  repeat  them 
here. 


544  CONNECTICUT    EXPERIMENT    STATION  BULLETIN   269. 

Perithecia,  however,  are  not  so  well  known.  Some  features 
noted  by  previous  observers  may  here  be  re-emphasized  and 
additional  ones  given.  Perithecia  have  been  found  by  the  writer 
in  roots  of  tobacco  (PI.  XXXVII,  Fig.  6),  the  pea  (PI.  XXXVII, 
Fig.  5),  the  violet  (PI.  XXXVII,  Figs.  2  and  4)  and  recently  of 
Antirrhinum.  Sections  have  been  made  of  the  first  three  infected 
roots  mentioned  and  the  perithecia  found  in  their  tissues  are  alike 
in  all.  They  are  found  chiefly  in  the  cortex,  but  they  are  also 
located  on  the  surface  lying  among  masses  of  chlamydospores 
and  no  tissue  of  the  root  is  entirely  free  from  their  invasion.  The 
description  of  them  as  seen  in  culture,  however,  applies  only  to 
those  isolated  from  the  violet  as  no  culture  of  them  has  been 
secured  from  any  other  host.  In  culture  the  perithecia  of 
Thielavia  basicola  Zopf  are  globular  or  nearly  so,  but  in  nature 
the  pressure  of  the  surrounding  cells  of  the  host  may  make 
them  some  what  elongated,  one  dimension  occasionally  being  nearly 
twice  as  great  as  the  other.  The  cells  of  the  tightly  twisted  and 
intertwined  hyphae,  composing  the  enveloping  sheath,  vary  in 
size  so  as  to  make  the  perithecium  "rough"  as  Gilbert  described  it. 
The  enveloping  sheath  is  hyaline  or  slightly  tinted.  At  maturity 
the  perithecium  looks  black  which  is  due  to  the  very  dark  brown 
color  of  the  ascospores  lying  within.  There  is  no  sign  of  an  opening 
of  any  kind  so  that  it  may  be  considered  a  true  cleistocarp.  The 
asci  are  exceedingly  fragile  and  by  the  time  the  ascospores  are 
mature  they  have  disintegrated  leaving  the  ascospores  free  in  the 
perithecial  cavity.  Asci,  however,  can  be  seen  and  obtained 
intact  by  gently  pressing  the  cover  glass  over  a  nearly  mature 
perithecium.  If  the  ascospores  are  mature  the  ascus  will  invariably 
be  ruptured.  The  asci  are  hyaline,  nearly  egg-shaped  and  contain 
eight  ascospores.     (PI.  XXXVIII,  Fig.  23.) 

The  size  of  the  perithecium  has  been  commonly  given  as  80  to 
100 /*.  The  writer  has  found  this  size  rather  large.  In  the  sections 
of  the  tobacco  and  pea  roots  the  perithecia  measure  about  the  same, 
the  maximum  size  of  those  measured  in  tobacco  being  72/*  and 
those  of  the  pea  66/*.  In  the  violet  roots  the  perithecia  were  some- 
what larger,  the  maximum  dimension  reaching  as  high  as  99/* 
which,  however,  is  exceptional  even  in  this  host.  The  larger  size 
of  the  perithecia  in  the  violet  roots  may  possibly  be  due  to  green- 
house conditions.  In  culture,  perithecia,  which  it  is  recalled  were 
isolated  from  the  violet,  average  about  the  same  in  size  as  those 
found  in  tobacco  and  pea  roots. 

The  ascospores  are  small,  dark  brown  and  lenticular  with  one  end 
blunt,  from  which  germination  occurs.  In  mass  they  appear  black. 
The  relatively  large  oil  drop  is  the  conspicuous  feature  of  the  cell 
contents.  In  the  roots  of  the  violet,  tobacco  and  pea,  as  well  as 
those  in  culture  1351,  the  general  size  is  10  to  13/*  by  4.5  to  6.5/*. 

The  ascogonium  begins  as  a  small  lateral  branch  attached  at 
right  angles  to  the  main  hypha.     The  early  stages,  in  gross  appear- 


PERITHECIA    OF    THIELAVIA   BASICOLA  545 

ance,  apparently  do  not  differ  essentially  from  the  figures  given  by 
Fraser  and  Chambers  (11)  for  Aspergillus  herbariorum  and  by  Dale 
(7)  for  Aspergillus  repens  de  Bary,  although  loose  coils  were 
not  observed  in  this  fungus.  No  attempt  has  been  made  to  study 
the  nuclear  behavior  and  all  the  drawings  of  these  stages,  given 
here  (PI.  XXXVIII,  Figs.  11-22)  were  made  from  living  material. 
The  coil  early  becomes  compact  and  additional  hyphae  soon  bud  out 
from  the  primary  coil,  thereby  concealing  it.  The  entire  process 
takes  place  very  quickly,  as  perithecia,  apparently  mature,  can  be 
seen  along  the  sides  of  the  tube  eight  days  after  the  transfer  has 
been  made.  Where  observed,  the  early  stages  gave  no  indication 
of  more  than  one  hypha  being  involved  in  the  ascogonial  coil. 

Possible  Factors  Inducing  the  Formation  of  Perithecia. 

Having  obtained  perithecia  in  this  accidental  way  the  question 
arose  as  to  the  factors  which  brought  forth  their  appearance.  It 
was  first  thought  that  this  might  be  a  specialized  form  growing  on 
this  particular  lot  of  violets.  More  violets  were  procured  from  the 
same  greenhouse  and  cultures  were  made  from  individual  plants. 
Ten  additional  cultures  were  isolated  from  as  many  different  plants, 
but  while  all  produced  endoconidia  and  chlamydospores,  none 
formed  perithecia.  If,  then,  culture  1351  were  a  unique  strain  it 
could  not  be  general  throughout  the  plants  in  the  greenhouse,  but 
one  might  expect  to  obtain  perithecia  from  one-spore  cultures  of  its 
endoconidia  and  chlamydospores.  For  the  purpose  of  investiga- 
ting this  point  one-endoconidium  cultures  were  made.  The  spores 
were  plated  in  stiff  agar  and  the  single  germinating  spores  were 
transferred  to  tubes. 

One-Conidium  Cultures. 

Of  the  260  one-endoconidium  cultures  that  were  isolated  from 
culture  1351  all  showed  a  luxuriant  development  of  endoconidia 
and  chlamydospores,  but  no  perithecia  were  found  in  them.  This 
result  apparently  gave  the  solution  as  to  the  cause  of  the  non- 
appearance of  perithecia  in  some  of  the  first  transfers  made  from 
culture  1351.  In  an  attempt  to  obtain  transfers  free  from  bacteria 
and  the  associated  fungus  an  effort  was  made  to  touch  the  mycelium 
very  lightly  with  the  needle  in  order  to  carry  over  only  endoconidia. 
For  a  time  it  was  thought  that  perithecia  were  completely  lost,  but 
later  it  was  found  that  transfers  which  included  some  of  the 
mycelium  always  produced  perithecia. 

In  isolating  the  germinating  endoconidia  it  was  noted  that  they 
vary  somewhat  in  shape  from  the  elongated  narrow  type  with 
straight  ends  to  short  almost  egg-shaped  ones.  The  one-spore 
cultures  were  made  from  as  many  different  types  as  possible,  but 
the  subsequent  growth  was  alike  in  all. 

Although  no  one-endoconidium  cultures  developed  perithecia 


546  CONNECTICUT    EXPERIMENT    STATION  BULLETIN   269. 

it  seemed  possible  that  culturing  two  or  more  together  might 
lead  to  their  production.  To  facilitate  the  process  of  culturing, 
transfers  from  several  tubes  were  frequently  put  into  one  tube 
so  that  while  all  the  260  cultures  were  used  only  151  mixed 
cultures  were  made.  In  case  perithecia  developed  in  a  tube  con- 
taining transfers  from  more  than  two  tubes  the  cultures  producing 
them  could  be  readily  separated.  No  perithecia  were  in  any  of 
these  tubes. 

Chlamydospore  Cultures. 

For  the  same  reason  chlamydospore  cultures  were  also  isolated 
from  culture  1351.  They  are,  however,  much  more  difficult  to 
secure  than  endoconidium  cultures,  but  by  treating  them  with 
pepsin  good  germination  was  obtained.  Pieces  of  culture,  three 
or  four  weeks  old,  were  thoroughly  washed,  since  the  numerous 
endoconidia,  which  are  everywhere  present  in  cultures,  germinate 
so  rapidly  as  to  easily  contaminate  the  chlamydospore  cultures. 
Chlamydospores  cling  tenaciously  together  in  chains  and  frequently 
more  than  one  chlamydospore  in  a  chain  will  germinate.  In  such 
cases  the  entire  chain  was  transferred  to  a  tube  with  the  intention 
of  accurately  making  single  spore  cultures  in  case  perithecia  were 
found  in  any  of  the  tubes.  Ninety  chlamydospore  cultures  were 
included  in  the  twenty  mixed  cultures  made,  each  tube  containing 
transfers  from  four  or  five  cultures,  but  in  none  of  these  twenty 
tubes  were  perithecia  produced.  It  was  the  intention  to  make  an 
extensive  series  of  plantings  of  one-endoconidium  cultures  with 
chlamydospore  cultures,  but  investigations  with  the  asco  stage 
made  this  seem  unnecessary  and  comparatively  few  were  made. 
In  no  case  did  these  produce  ascospores  but  both  asexual  spores. 

Investigations  with  the  Ascospore  Stage. 

In  studying  the  young  stages  of  the  ascogonium  an  effort  was 
made  to  ascertain  the  relation  of  the  ascogonial  hypha  to  the 
mycelium  which  bears  endoconidia  and  chlamydospores,  since  as 
noted  above,  culture  1351  is  a  mixed  culture  bearing  all  three  spore 
forms.  As  the  figures  of  Aspergillus  also  show,  there  was  no 
indication  here  of  hyphae  from  two  different  mycelia  being  con- 
cerned in  the  ascogonial  coil.  With  the  hope  of  getting  a  clearer 
view  of  the  individual  threads  and  also  of  being  able  to  get  cultures 
from  them,  small  pieces  of  mycelium  were  cultured  on  clear  agar 
with  the  idea  that  possibly  an  ascogonial  hypha  might  become 
sufficiently  separated  so  that  it  could  be  cut  free  from  contam- 
inating endoconidia,  chlamydospores  or  the  mycelium  which  bears 
them.  All  such  attempts  failed  and  one-ascospore  cultures  seemed 
to  be  the  only  method  of  getting  accurate  evidence  of  the  relation- 
ship of  the  perithecia  to  the  endoconidia  and  chlamydospores. 


PERITHECIA    OF    THIELAVIA    BASICOLA  547 

The  Germination  of  Ascospores.  Endoconidia  are  produced 
in  such  numbers  that  it  is  also  necessary  to  free  perithecia  from 
them  in  order  to  procure  pure  ascopore  cultures,  for  they  germ- 
inate so  readily  that  they  contaminate  the  smaller  and  more 
slowly  germinating  ascospores.  Pieces  of  culture,  containing 
many  perithecia,  were  thoroughly  washed  under  a  strong  flow  of 
tap  water  and  finally  in  sterilized  water.  It  was  often  necessary 
to  tease  away  the  endoconidia  with  sterilized  needles. 

Various  methods,  recommended  for  inducing  germination  of 
other  ascospores,  were  tried  without  success.  However,  in  the 
writer's  experience  the  germination  of  ascospores  of  Thielavia 
basicola  is  very  low,  so  this  may  be  the  cause  of  the  failure.  The 
first  germination  was  obtained  in  a  van  Tieghem  cell  containing 
a  trace  of  pepsin  in  the  drop  of  water.  All  subsequent  cultures 
were  made  by  crushing  the  washed  perithecia  in  a  tube  containing 
beef  broth,  a  trace  of  pepsin  and  a  minute  particle  of  thymol. 
A  better  method,  probably,  would  be  to  filter  the  solution  con- 
taining pepsin  through  a  Berkefeld  filter,  thus  making  thymol 
unnecessary.  After  about  half  an  hour  loops  of  the  broth  were 
transferred  to  melted  beef  agar  which  contained  about  3%  agar. 
A  very  clear  agar  is  essential  on  account  of  the  small  size  of  the 
ascospores.  Germination  begins  to  appear  in  about  five  days. 
The  open  Petri  dish  was  examined  under  the  microscope  and 
germinating  ascospores  transferred  with  a  sterile  needle  to  a  suit- 
able medium,  in  this  case  pea  meal  agar.  By  this  method  forty- 
two  one-ascospore  cultures  were  obtained.  The  germination  of 
these  spores  was  obtained  only  when  pepsin  was  used  and  it  was 
not  determined  whether  or  not  they  will  germinate  without  it. 
The  sole  aim  was  to  procure  germination  and  no  consideration 
was  given  to  factors  bringing  it  about.  It  is  quite  possible  that 
freezing  may  be  an  aid  to  germination  of  the  ascospores  and  the 
age  of  the  spores  may  also  be  important.  The  best  germination 
was  secured  with  spores  taken  from  cultures  six  weeks  or  two 
months  old. 

As  previously  stated  the  ascospore  germinates  at  the  blunt  end 
(PI.  XXXVIII,  Fig.  1)  and  the  writer  has  not  found  more  than 
one  germ  tube  connected  with  a  spore.  Next  to  the  spore  coat, 
the  germ  tube  has  a  one-sided  bulbous  enlargement  and  variation 
in  diameter  with  considerable  curling  is  characteristic  of  the  older 
as  well  as  the  younger  hyphae  (PL  XXXVIII,  Figs.  9,  10). 
Especially  in  the  early  stages  the  cells  are  practically  filled  with 
a  fine  granular  content  (Pl.XXXVIII,  Figs.  1-8).  Growth  is  com- 
paratively slow  and  although  small  test  tubes  were  used  it  was 
several  days  before  the  delicate  hyphae  could  be  detected  growing 
up  on  the  sides  of  the  tube  and  along  the  edge  of  the  medium. 
The  mycelium  is  white  and  on  pea  meal  agar  seldom  shows  the 
slightest  tendency  to  become  cottony,  neither  does  it  make  a  felt- 
like growth  as  does  Thielaviopsis  basicola.  The  hyphae  grow 
sparsely  over  the  surface  and  down  into  the  interior  of  the  medium. 


548  CONNECTICUT    EXPERIMENT    STATION  BULLETIN   269. 

None  of  the  forty-two  one-ascospore  cultures  produced  either 
chlamydospores  or  endoconidia  and  although  they  have  been 
in  culture  more  than  two  years  they  have  produced  only  perithecia 
and  these  usually  developed  sparingly  and  sometimes  not  at  all. 
While  there  may  be  some  difference  of  growth  in  the  media  com- 
monly used  for  fungi,  there  has  never  been  the  least  indication  of 
even  the  rudiments  of  chlamydospores  or  endoconidia.  Pea  meal 
agar  is  the  stock  medium  used  and,  while  the  growth  on  it  is  usually 
so  delicate  as  to  be  readily  overlooked,  it  seems  to  be  a  satisfactory 
one  for  this  fungus,  as  all  the  forty-two  cultures  are  still  in  good 
condition.  The  fungus  also  grows  well  on  corn  meal,  prune,  oat, 
potato,  carrot  and  bean  meal  agars.  However,  there  is  often  a 
difference  in  growth  of  cultures  made  at  the  same  time  and  on 
the  same  agar.  The  factor,  or  factors,  bringing  about  this  condi- 
tion have  not  been  ascertained.  With  the  above  media  in  only  a 
few  cultures  was  there  any  approach  to  a  cottony  mycelium  and, 
while  the  mycelium  was  abundant,  it  still  remained  delicate,  being, 
in  comparison  with  other  fungi,  verv  inconspicuous. 

Mixed  Cultures  of  Thielavia  basicola  Zopf  and 
Thielaviopsis  basicola  (Berk.)  Ferraris 

The  fact  that  none  of  the  one-ascospore  cultures  produced  either 
endoconidia  or  chlamydospores  suggested  that  the  ascospore 
cultures  might  be  sexually  related  to  the  endoconidium-chlamy- 
dospore  strains  without,  however,  producing  any  asexual  spores. 
With  this  idea  in  mind  each  of  three  one-ascospore  cultures  was 
cultured  with  eighty-one  one-endoconidium  cultures,  from  culture 
1351,  and  with  three  Thielaviopsis  basicola  from  tobacco,  four 
from  violet  and  one  from  garden  peas.  Perithecia  were  abundantly 
produced  in  all.  Usually  within  six  days  immature  perithecia 
could  be  detected  in  the  tube  placed  under  the  microscope  and  in 
eight  days  almost  mature  perithecia  could  be  seen  with  a  hand  lens. 
The  remaining  thirty-nine  perithecial  cultures  were  cultured  with 
the  eight  cultures  isolated  from  tobacco,  violet  and  pea  and  they 
were  also  stimulated  to  produce  an  abundance  of  perithecia. 

These  results  seemed  to  indicate  that  production  of  perithecia  in 
this  fungus  was  due  to  different  sexual  strains.  However,  it  was 
early  noted  that  there  was  a  tendency  to  form  perithecia  in  the 
one-ascospore  cultures  even  though  they  were  not  cultured  with 
the  endoconidium-chlamydospore  strains.  In  such  cases  perithecia 
were  slower  in  making  their  appearance  and  increased  in  number 
as  the  culture  grew  older.  Furthermore  the  forty-two  cultures 
varied  somewhat  in  this  ability  of  forming  perithecia  when  cul- 
tured alone  and  there  was  often  a  difference  in  cultures  of  the 
same  strain  made  at  the  same  time  and  on  the  same  medium.  In 
no  case,  however,  did  a  single  culture  alone  produce  perithecia 
as  quickly  or  as  abundantly  as  when  grown  with  endoconidium- 


PERITHECIA    OF    THIELAVIA    BASICOLA  549 

chlamydospore  cultures.  The  factors  which  cause  this  difference 
in  production  of  perithecia  have  not  been  ascertained.  In  auto- 
claves in  which  steam  is  in  contact  with  the  tubes  a  little  more 
moisture  at  times  collects  in  some  tubes  than  in  others,  so  it  was 
thought  that  possibly  the  rigidity  of  the  medium  might  be  a  factor. 
Cultures  were  made  on  media  containing  different  concentrations 
of  agar,  but  these  gave  no  definite  results,  perithecial  formation 
not  being  increased  in  any  of  them.  A  few  cultures  on  oat  agar, 
made  in  the  usual  way,  formed  a  ring  of  perithecia  along  the  upper 
edge  of  the  medium,  but  these  were  not  general  throughout  the 
tube  and  not  always  obtained.  Possibly  a  slow  drying  combined 
with  a  favorable  medium  might  be  a  factor.  Occasionally  transfers 
taken  from  an  old  dried  culture  make  a  more  luxuriant  growth  and 
form  more  perithecia  than  those  from  newer  cultures.  For  the 
experiments  made  on  the  production  of  perithecia  an  ascosporic 
culture.  No.  1603,  which  alone  seldom  forms  perithecia,  was  used. 

Mixed  Cultures  of  Thielavia  basicola  Zopf  and  other 

Fungi. 

The  writer  is  indebted  to  Prof.  Thaxter  for  the  suggestion  of 
culturing  Thielavia  basicola  Zopf  with  other  fungi  to  see  if  the 
reaction  with  them  is  similar  to  that  with  Thielaviopsis  basicola 
(Berk.)  Ferraris. 

First  of  all,  however,  Thielavia  basicola,  culture  1603,  the  strain 
which  alone  rarely  produces  perithecia,  was  cultured  with  the 
remaining  forty-one  strains  of  Thielavia  basicola,  but  there  was  no 
additional  stimulation  in  growth  or  perithecial  formation.  In  this 
respect  the  results  differ  from  those  obtained  by  Miss  Wineland 
(32)  who  found  that  two  one-ascospore  cultures  of  Fusarium 
moniliforme,  grown  together,  stimulate  the  production  of  perithecia. 

Other  fungi  were  then  tried.  The  same  Thielavia  basicola  cul- 
ture, No.  1603,  was  cultured  with  120  strains  and  species,  embrac- 
ing 43  genera,  belonging  to  Phycomycetes,  Ascomycetes,  Basi- 
diomycetes  and  Fungi  Imperfecti.  In  all  the  tests  pea  meal 
agar  was  used  and  in  each  case  a  transfer  from  one  culture 
of  Thielavia  basicola  was  put  into  a  tube  with  that  from  one  other 
fungus.  Many  of  the  fungi  grew  so  rapidly  as  to  bury  the  delicate 
more  slowly  growing  Thielavia  basicola  and  no  evidence  of  its 
mycelium  could  be  detected,  but  in  others  both  species  survived 
in  the  fully  mature  culture. 

As  a  result  of  these  experiments  it  was  found  that  Thielavia 
basicola  Zopf  produces  abundant  perithecia  when  it  is  cultured 
with  Cladosporium  fulvum,  Aspergillus  umbrosus,  a  strain  of  Asper- 
gillus glaucus,  Eurotium  amstelodami  and  in  a  less  degree  with 
Fusicladium  pirinum.  In  all  cases  the  perithecia  produced  in 
these  mixed  cultures  are  exactly  alike  in  every  respect  to  those 
obtained  in  the  original  culture  isolated  from  violet  and  no  trace  of 
chlamydospores  or  endoconidia  was  found  in  them. 


550  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    269. 

It  seems  probable  that  if  various  other  media  were  used  or  change 
of  cultural  conditions  tried  that  other  fungi  might  react  in  this 
same  way. 

It  is  interesting  that  results  vary  with  different  species  of  the 
same  genus.  While  perithecia  of  Thielavia  basicola  were  abund- 
antly produced  when  that  fungus  was  cultured  with  Cladosporium 
julvum,  isolated  from  tomato  leaf  by  the  writer,  three  Cladosporium 
cultures  received  from  Dr.  C.  L.  Shear,  two  of  which  were  isolated 
from  cranberry  and  one  from  blueberry,  were  unsuccessful. 
Aspergillus  umbrosus,  kindly  determined  by  Drs.  Charles  Thorn 
and  Margaret  Church,  was  obtained  from  peach  preserves  and 
1  Aspergillus  glaucus  strain  and  Eurotium  amstelodami  were  received 
from  the  same  investigators.  Eight  additional  cultures  of  Aspergil- 
lus, received  from  them  and  nine  others  of  the  same  genus,  received 
from  Dr.  D.  H.  Linder,  as  well  as  one  other  species  from  this  lab- 
oratory, had  no  effect  in  stimulating  the  production  of  perithecia. 
Thielaviopsis  paradoxa  has  a  striking  resemblance  to  Thielaviopsis 
basicola,  but  two  cultures  received  from  Dr.  J.  P.  Martin,  Honolulu, 
and  one  isolated  from  banana  stalk  by  the  writer,  were  also 
unsuccessful.  Two  cultures  of  Isaria,  received  from  Dr.  F.  J. 
Pritchard,-had  no  effect  in  increasing  the  number  of  perithecia. 

Results,  similar  to  those  given  above,  are  recorded  by  Heald 
and  Pool  (13)  who  found  that  Melanospora  pampeana  responded 
in  the  production  of  perithecia  when  grown  with  Fusarium  monili- 
jorme,  Basisporium  gallarum  and  to  a  smaller  extent  with  Fusarium 
culmorum.  Their  description  of  a  one-ascospore  culture  of  Melano- 
spora pampeana  as  showing  a  "scanty  scarcely  distinguishable, 
white  mycelium"  might  apply  equally  as  well  to  the  one-ascospore 
cultures  of  Thielavia  basicola  Zopf.  However,  in  their  one-asco- 
spore culture  or  in  transplants  from  it  they  never  found  perithecia, 
while  on  the  other  hand  the  one-ascospore  cultures  of  Thielavia 
basicola  Zopf  spasmodically  produce  some  perithecia  when  grown 
alone  on  an  ordinary  culture  medium.  In  opposition  to  the  results 
of  Heald  and  Pool  the  writer  so  far  has  not  been  able  to  get  a 
response  of  perithecia  of  Thielavia  basicola  on  cultures  of  other 
fungi  that  have  been  heated  in  the  autoclaye  or  boiled  in  the  open 
for  twenty  minutes. 

Molliard  (16),  (17)  found  that  Ascobolus  produces  perithecia 
when  a  bacterium  is  present  in  the  culture  and  Sartory  announced 
a  similar  association  necessary  for  the  production  of  ascospores  in 
a  yeast  (25)  and  of  perithecia  in  an  Aspergillus  (26),  (27),  (28). 

The  writer's  experiments  have  been  sufficiently  repeated  to 
demonstrate  that  Thielavia  basicola  Zopf,  though  possessing  to 
some  degree  the  ability  to  form  perithecia  when  cultured  alone,  is 
greatly  stimulated  in  the  production  of  these  structures  when 
cultured  in  association  with  Thielaviopsis  basicola  (Berk.)  Ferraris, 
Cladosporium  julvum,  Aspergillus  umbrosus,  Aspergillus  of  the 
glaucus  group,  Eurotium  amstelodami  and  Fusicladimu  pirinum. 


PERITHECIA    OF    THIELAVIA    BASICOLA  551 

Since  Thielavia  basicola  Zopf  does  not  form  either  the  chlamydo- 
spores  or  endoconidia  of  Thielaviopsis  basicola  (Berk.)  Ferraris, 
and  since  perithecial  formation  is  stimualted  equally  well  by  other 
fungi,  there  seems  to  be  no  real  justification  for  considering 
Thielavia  basicola  the  perfect  stage  of  Thielaviopsis  basicola. 
Although  the  frequent  association  of  these  two  fungi  in  nature 
and  the  fact  that  their  relationship  has  long  been  accepted  in 
literature  make  one  reluctant  to  consider  them  as  two  distinct 
species,  yet  the  cultural  evidence  is  a  strong  argument  in  favor  of 
such  a  view. 

The  Influence  of  Extracts  upon  Production  of  Perithecia. 

The  observations  made  upon  the  influence  of  extracts  of  some 
fungi  upon  production  of  perithecia  are  recent,  but  the  experiments 
have  been  repeated  a  sufficient  number  of  times  to  make  them 
seem  worthy  of  being  recorded  here.  Cultures  of  several  fungi 
were  barely  covered  with  water  and  allowed  to  stand  about 
twenty-four  hours.  The  liquid  was  filtered  through  paper  and 
then  through  a  Berkefeld  filter,  thus  avoiding  sterilization  by  heat; 
for,  as  already  stated,  cultures  of  the  various  fungi,  which  in  living 
condition  stimulate  the  production  of  perithecia,  after  heating,  no 
longer  have  this  stimulating  effect.  In  some  cases  the  liquid  only 
was  used  and  in  others  the  mat  of  mycelium  was  crushed  in  a 
mortar,  squeezed  through  cheese  cloth  and  the  juice  thus  obtained 
also  filtered  with  the  extract.  There  is  some  indication  that  the 
latter  method  is  the  better  one,  but  this  point  has  not  been  fully 
determined.  All  the  extracts  from  fresh  fungi,  so  far  tried,  have 
been  made  with  water  only  and  sterilized  by  passing  the  liquid 
through  a  Berkefeld  filter.  Cultures  of  Thielavia  basicola  Zopf, 
No.  1603,  were  made  in  the  usual  way  upon  pea  meal  agar  and 
some  of  the  extract,  prepared  as  above  described,  poured  into  the 
tube  and  allowed  to  flow  over  the  transplant.  In  every  case  the 
fungus  from  which  the  extract  was  made  was  also  grown  upon 
pea  meal  agar  to  avoid  conflicting  results  which  might  arise  from 
the  introduction  of  some  other  medium.  Check  cultures  on  pea 
meal  agar  alone  were  also  made  in  every  series. 

Nine  different  extracts  of  Thielaviopsis  basicola  (Berk.)  Ferraris 
were  made  and  each  one  caused  a  marked  stimulation  both  in 
growth  and  perithecial  production,  in  the  majority  of  cases  fully 
equaling  in  numbers  the  perithecia  formed  in  mixed  cultures  of 
the  two  living  fungi.  Following  the  use  of  extracts  a  stimulation 
in  growth  is  noticeable  within  a  few  days,  but  the  formation  of 
perithecia  is  slower  than  when  two  living  fungi  are  cultured 
together.  However,  in  every  series,  the  checks  have  no  perithecia 
or  only  a  few  scattered  ones,  while  the  cultures  treated  with  the 
extracts  have  produced  them  abundantly.  The  age  of  the  fungus 
from  which  the  extract  is  made,  amount  of  water  necessary  for 


552  CONNECTICUT    EXPERIMENT    STATION  BULLETIN    269. 

extracting  and  the  optimum  length  of  time  required  for  extraction 
are  probable  factors  which  affect  the  number  and  rapidity  of 
perithecial  development. 

Three  different  extracts  were  made  from  Cladosporium  fulvum 
and  one  from  Aspergillus  umbrosus.  In  all  cases  these  extracts 
also  greatly  stimulated  growth  and  perithecial  production.  As 
when  grown  with  the  living  fungi,  Thielavia  basicola  produces  no 
asexual  spores  when  treated  with  any  of  the  extracts  so  far  used. 

The  effect  of  heat  upon  the  extracts  was  tried  both  in  the  auto- 
clave at  twenty  pounds  pressure  for  twenty  minutes  and  by  boiling 
in  the  open  for  twenty  minutes.  Without  exception  both  methods 
of  heating  have  so  far  completely  destroyed  the  stimulating  power 
of  the  extract.  Extracts  from  Aspergillus  of  the  glaucus  group, 
Eurotium  amstelodami  and  Fusicladium  pirinum  have  not  yet  been 
tried. 

As  stated  above,  Thielaviopsis  paradoxa,  in  living  condition, 
failed  to  stimulate  the  production  of  perithecia  and  it  was  also 
suggested  that  the  failure  with  many  fungi  in  living  condition 
might  be  due  to  the  smothering  of  the  more  delicate  Thielavia 
basicola.  If  this  is  the  situation,  then,  extracts  from  such  luxuriant 
fungi  might  act  as  stimulants  even  though  the  living  fungus  gave 
no  result.  While  one  experiment  is  not  sufficient  to  be  conclusive 
it  may  be  stated  here  that  an  extract  made  from  Thielaviopsis 
paradoxa  also  greatly  stimulated  growth  and  perithecial  formation. 
The  case  is  the  same  with  Saccharomyces  cerevisiae.  Cultures  of 
yeast  were  made  on  pea  meal  agar  and  mixed  cultures  were  made 
from  them  and  Thielavia  basicola.  In  each  case  the  yeast  com- 
pletely covered  the  Thielavia  basicola  transplant  before  it  had 
time  to  grow;  but  two  different  extracts  made  from  fresh  yeast 
cultures,  stimulated  growth  and  perithecial  production.  The 
stimulating  factor  in  the  yeast  extract  was  also  completely 
destroyed  by  heating. 

To  determine  whether  commercial  extracts  of  fungi  contained 
the  stimulating  factor,  1%,  2%,  4%  and  6%  water  solutions  of 
Taka-diastase  were  made  and  filtered  through  a  Berkefeld  filter. 
Cultures  of  Thielavia  basicola  were  treated  with  these  by  the  same 
method  as  with  the  preceding  extracts.  The  resulting  growth  far 
surpassed  that  following  the  use  of  any  other  extract  so  far  used. 
The  mycelium  became  very  abundant  and  made  almost  a  felt-like 
growth.  Later,  perithecia  developed.  The  type  of  growth  follow- 
ing the  addition  of  Taka-diastase  differs  so  markedly  from  that  in 
any  medium  or  extract  which  has  been  tried  that  it  suggests  the 
presence  of  two  factors,  one  an  unusually  potent  stimulant  for 
growth  and  another  stimulating  perithecial  formation.  See  Plate 
XXXIX. 

The  nature  of  the  factors  in  any  of  the  extracts  above  considered, 
causing  such  a  striking  increase  in  growth  and  production  of 
perithecia  of  Thielavia  basicola,  is  beyond  the  scope  of  the  problem 
at  hand.  The  results  with  the  mixed  cultures  of  Thielavia  basicola 
Zopf  and  of  other  fungi  are  an  indication  that  this  fungus  should 


PERITHECIA    OF    THIELAVIA    BASICOLA  553 

not  be  considered  the  perfect  stage  of  Thielaviopsis  basicola  (Berk.) 
Ferraris  any  more  than  of  the  other  fungi  above  noted.  The 
results  with  extracts,  here  recorded,  are  an  additional  indication 
that  Thielavia  basicola  is  a  distinct  fungus  and  they  are  also  an 
argument  against  any  ideas  of  heterothallism  that  may  have  been 
connected  with  it. 

Thielavia  basicola  Zopf  has  always  been  found  associated  with 
Thielaviopsis  basicola  (Berk.)  Ferraris  on  the  same  hosts.  Whether 
or  not  it  is  by  itself  parasitic  on  these  hosts  or,  as  has  been  suggested 
by  Dr.  Clinton,  a  parasite  on  Thielaviopsis  basicola,  is  not  yet 
determined.  Inoculations  of  Thielavia  basicola  have  been  made  on 
tobacco  in  the  greenhouse  as  well  as  in  the  field  without  definite 
results.  The  biologic  relationship  of  these  two  forms  is  not  yet 
clear. 

Summary. 

Perithecia  of  Thielavia  basicola  Zopf  have  been  secured  in 
artificial  culture. 

Since  the  name  Thielavia  basicola  was  given  by  Zopf  on  account 
of  the  presence  of  perithecia,  the  fungus  secured  from  their  asco- 
spores  should  be  considered  Thielavia  basicola  Zopf.  This  is  the 
first  report  of  its  isolation  in  artificial  culture. 

Thielavia  basicola  Zopf  has  a  tendency  to  produce  perithecia 
when  grown  alone,  but  this  is  greatly  stimulated  when  the  fungus 
is  grown  with  Thielaviopsis  basicola  (Berk.)  Ferraris  with  which 
it  is  associated  in  nature. 

It  has  been  shown  that  Thielavia  basicola  Zopf  is  also  stimulated 
to  produce  perithecia  when  it  is  grown  with  Cladosporium  fulvum, 
Aspergillus  umbrosus,  Aspergillus  of  the  glaucus  group,  Eurotium 
amstelodami  Mangin,  and  to  a  certain  extent  with  Fusicladium 
pirinum. 

It  has  been  shown  that  Thielavia  basicola  Zopf  is  likewise  stimu- 
lated to  produce  perithecia  when  it  is  treated  with  water  extracts 
obtained  from  Thielaviopsis  basicola  (Berk.)  Ferraris,  Cladosporium 
fulvum,  Aspergillus  umbrosus,  Thielaviopsis  paradoxa,  Saccharo- 
myces  cerevisiae  and  also  with  a  water  solution  of  Taka-diastase. 

The  above  evidence  indicates  that  Thielavia  basicola  Zopf  is 
not  the  ascospore  stage  of  Thielaviopsis  basicola  (Berk.)  Ferraris 
although  the  two  forms  are  commonly  associated. 

Literature  Cited. 

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4.  Cappelluti-Altomare,  G.    I  semenzai  del  Tabacco  e  la  Thielavia  basi- 

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Compt.  Rend.  Soc.  Biol.    72:  558-560.     1912. 

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27.  Sartory,  A.    Sporulation  by  symbiosis  in  fungi.    Compt.  Rend.  Acad. 

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Explanation  of  Plates. 

Plate  XXXVII. 

Fig.  1.  Violet  plants  infected  with  Thielaviopsis  basicola  (Berk.)  Fer- 
raris and  Thielavia  basicola  Zopf. 

Fig.  2.  Part  of  section  of  infected  violet  root.  p,  perithecia  of  Thielavia 
basicola. 

Fig.  3.  Part  of  section  of  infected  violet  root  showing  sclerotial  masses 
of  hyphae  and  chlamydospores  of  Thielaviopsis  basicola. 
Photographed  by  Mr.  E.  M.  Stoddard. 

Fig.  4.  Part  of  section  of  infected  violet  root  showing  perithecia  of 
Thielavia  basicola. 

Fig.  5.  Part  of  section  of  infected  pea  root  showing  perithecia  of  Thie- 
lava  basicola.     Photographed  by  Mr.  E.  M.  Stoddard. 

Fig.  6.  Part  of  section  of  infected  tobacco  root  showing  perithecia  of 
Thielavia  basicola.      Photographed  by  Mr.  E.  M.  Stoddard. 

Plate  XXXVIII. 

Figs.  1—8.  Germination    of   ascospores    of    Thielavia  basicola    Zopf. 

Figs.    1   and  6   magnification   x   1,000;   Figs.   2-5   and 
7-8  mag.  x  450. 

Figs.  9  and  10.      Hyphae  of    Thielavia  basicola  Zopf.  x  600. 

Figs.  11-22.  Stages  in  the  development  of  the  perithecium  of    Thie- 

lavia basicola  Zopf.      Figs.   11-16,  18,  20-22,  magnifi- 
cation x  1200.      Figs  17  and  19  mag.  x  1,000. 

Fig.  23.  An    ascus    of     Thielavia  basicola   Zopf    with    ascospores. 

x  1,200. 


PLATE  XXXVII. 


- 


:. 


Sc.Mgl 


4 


.+* 


^5® 


■^r 


PLATE  XXXVIII. 


PLATE  XXXIX 


Cultures  of  1603  on  pea  meal  agar.      P,  perithecia. 
No.  1.      Addition  of  a  few  drops  of  extract  from  Cladosporium  fulvum. 
No.  2.      Addition  of  a  few  drops  of  extract  from    Thielaviopsis  paradoxa. 
No.  3.     Addition  of  a  few  drops  of  6%  water  solution  of  Taka-diastase. 

Check.      Pea  meal  agar  alone. 

No.  4.     Addition  of  a  few  drops  of  extract  from    Thielaviopsis  basicola 
(Berk.)  Ferraris. 

No.  5.      Addition  of  a  few  drops  of  extract  from  Saccharomyces  Cerevisiae. 

No.  6.     Addition  of  a  few  drops  of  extract  from  Aspergillus  umbrosus. 

Photographed  by  E.  M.  Stoddard. 


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